Simple, rapid spectrophotometric, and reverse-phase high performance liquid chromatographic methods were developed for the concurrent analysis of 17-beta-estradiol (ESR) and drospirenone (DRS). The spectrophotometric method was based on the determination of first derivative spectra and determined ESR and DRS using the zero-crossing technique at 208 and 282 nm, respectively, in methanol. The linear range was 0.5–32.0
Drospirenone (DRS), chemically (6R,7R,8R,9S,10R,13S,14S,15S,16S,17S) 1,3′,4′,6,6a,7,8,9,10,11,12,13,14,15,15a,16-hexadecahydro-10, 13-dimethylspiro-[17H-dicyclopropa [6,7:15,16]cyclopenta[a]phenanthrene-17, 2′(5′H)-furan]-3,5′(2H)-dione (Figure
Molecular structure of drospirenone (a) and 17
17
So far, some high performance liquid chromatograph (HPLC) techniques coupled with ultraviolet (UV) [
A number of efficient analytical techniques and procedures have been developed for the determination and pharmacokinetic studies of ESR individually as well as in combination with other drugs in pharmaceutical formulations, biological matrices, nutrients, and in
To the best of our knowledge, the simultaneous determination of ESR and DRS with the HPLC-UV method and spectrophotometric method has not yet been reported in the literature. The purpose of this study was to develop and validate an easy, precise, and selective RP-HPLC and derivative spectrophotometric method for the simultaneous determination of drugs in bulk and in tablets.
Spectrophotometric measurements were carried out with a Shimadzu UV-160 double beam spectrophotometer. Analysis was performed on the following operating conditions: 1-cm path length quartz cells, high scan speed, scan range 200–400 nm, 2 nm of slits width, and derivatives interval (Δ
HPLC measurements were performed on the Thermo Separation system (San Jose, CA) with the following parts: controller SN 4000, pump P 4000 and auto sampler AS 3000, fitted with 20
Separation on a Waters Symmetry C18 column (4.6 mm × 250 mm, in diameter 5
Drospirenone (DRS) and 17
Stock solutions of the studied drugs at 1.0 mg·mL−1 were prepared separately in methanol. The preparations of working standard solutions were made by appropriate dilutions from stock solution in methanol for the spectrophotometric methods and with acetonitrile-water (70 : 30, v/v) for the HPLC method.
Aliquots of standard solution of ESR and DRS (each 0.1 mg·mL−1) in mixture were transferred into 10 mL volumetric flasks to obtain the final concentrations of 0.5–8
The zero order and first order derivative absorption spectra of standard solutions in the range of 200–400 nm were recorded against a blank solvent. Firstly, the zero order spectra were recorded and then they transformed into their first derivative order form. Zero-crossing amplitudes in the first order derivative spectra were measured at 208 and 282 nm for ESR and DRS, respectively. Each concentration level was performed using 6 independent assays. To determine the calibration curves, the first order derivative amplitude values of each compound were plotted against the concentrations and the corresponding regression equations were obtained.
The standard solutions of ESR and DRS in the mixture at six different concentration levels were transferred into 10 mL volumetric flasks to achieve final concentrations of 0.23–7.5
Five tablets were weighed and finely powdered. The powder equivalent to an average tablet was weighed and then transferred to a 50 mL volumetric flask with 30 mL methanol and sonicated at room temperature for 1 h. The volume was completed with methanol and filtered. Tablet solution was appropriately diluted with methanol for the derivative spectrophotometric method and with acetonitrile : water (70 : 30, v/v) for the HPLC method. The solutions were then determined under specified conditions as in the section “general procedures and calibration curves.” Corresponding amounts of the drugs in the tablets were analyzed by related regression equations of the calibration curves.
Direct UV-absorption method was found to be inappropriate for the simultaneous determination of ESR and DRS due to some spectral interference. In addition, the wavelength of absorbance of ESR was lower than 205 nm and gave absorption bonds that were not sharp enough especially at low concentrations (Figure
Absorption spectra of ESR and DRS in methanol (both are 5
However, derivative spectrophotometry which is based on mathematical transformation has the advantages of reducing background absorbance and increasing the resolution of overlapping spectral bands and allows for the simultaneous analysis of organic compounds in the mixtures. Other important advantages of derivative spectroscopy are suppressing broad bands relatively to sharp bands and developing spectral details.
For the reasons described hereinabove, the derivative spectra of ESR and DRS solutions from first up to fourth were recorded separately and their spectra were compared in a row by memory of the device. The 1st order derivative (1D) spectroscopy was chosen for simultaneous determination due to the obtained zero crossing points for both compounds. The optimum wavelength without interferences for EST and DRS was 208 and 282 nm, respectively (Figure
First order derivative absorption spectra of ESR (
For the derivative UV spectrophotometric method, methanol and acetonitrile alone and with mixtures of 50% water were tested as the solvent and methanol was found to be the most suitable solvent by considering the sensitivity, noise level, and resolution.
An RP-HPLC method has also been developed for the simultaneous determination of ESR and DRS. In order to improve the resolution of the drugs, methanol-water and acetonitrile-water in different portions were tested as the mobile phase. The best results in terms of obtaining sharp peaks, resolution, and analysis time were obtained using acetonitrile : water (70 : 30, v/v). A Phenomenex C18-column, Venusil XBP C18 (Agela), and a Waters Symmetry C18-column were tried to obtain the best separation. Waters Symmetry C18-column was selected for the accurate quantitation of both drugs. The optimized detection wavelengths and flow rate were 279 nm and 1 mL/min, respectively, at room temperature. The average retention time of the ESR and DRS was approximately 3.54 and 4.55 min, respectively. RSD% of the retention times for both drugs was approximately 2.18% for 9 independent analyses. A typical chromatogram of drugs in mixture in selected conditions is shown in Figure
Schematic representation of chromatogram of ESR and DRS in selected conditions (both are 1.70
Calibration curves parameters were summarized in Table
Results of analytical parameters of proposed methods.
Parameters | Derivative spectrophotometric method | HPLC method | ||
---|---|---|---|---|
ESR | DRS | ESR | DRS | |
Linearity range ( |
0.5–8.0 | 0.5–32.0 | 0.23–7.5 | 0.08–2.5 |
Regression equationa | ||||
Slope | 0.014 | 0.003 | 45.83 | 264.95 |
Intercept | 0.009 | 0.001 | 1.23 | 2.208 |
Correlation coefficient ( |
0.9967 | 0.9998 | 0.9999 | 1.0 |
SD of |
0.000 | 0.000 | 0.842 | 2.86 |
SD of |
0.000 | 0.000 | 0.71 | 1.43 |
LOD ( |
0.14 | 0.10 | 0.05 | 0.02 |
LOQ ( |
0.42 | 0.29 | 0.15 | 0.05 |
a
Limit of detection (LOD) and quantification (LOQ) of drugs for proposed methods was calculated with the following equation: LOD = 3.3
Intraday and interday accuracy and precision were validated by solutions of drugs at three different concentrations for both proposed methods. Determinations were performed at five replicates within the same day for intraday and on five separate days for interday precision. For intraday and interday precision, the percent relative standard deviation (RSD%) values of ESR ranged from 0.01 to 0.32% and 0.45 to 1.07%, respectively, for the derivative spectroscopy method (Table
The intraday and interday accuracy and precision analysis of ESR and DRS by derivative spectrophotometric and HPLC methods (
Method | Drug | Concentration |
Intraday |
Interday |
---|---|---|---|---|
Derivative spectrophotometric method | ESR | 0.63 | 0.63 ± 0.32 | 0.56 ± 1.07 |
2.50 | 2.44 ± 0.01 | 2.38 ± 0.50 | ||
5.00 | 5.06 ± 0.01 | 4.94 ± 0.45 | ||
DRS | 1.00 | 0.89 ± 1.35 | 0.88 ± 2.39 | |
5.00 | 5.01 ± 1.94 | 4.93 ± 2.84 | ||
16.00 | 16.31 ± 1.04 | 16.28 ± 1.17 | ||
|
||||
HPLC method | ESR | 0.63 | 0.62 ± 1.18 | 0.64 ± 1.03 |
1.25 | 1.26 ± 0.21 | 1.26 ± 0.79 | ||
5.00 | 5.00 ± 0.02 | 5.01 ± 0.08 | ||
DRS | 0.16 | 0.16 ± 0.69 | 0.15 ± 2.67 | |
0.63 | 0.63 ± 1.90 | 0.62 ± 2.58 | ||
1.25 | 1.26 ± 1.75 | 1.24 ± 0.15 |
Recovery studies were conducted by spiking known amounts of pure compounds solutions at three different concentrations to a known amount of tablet solutions.
The results given in Table
Recovery of ESR and DRS determined by the proposed methods (
Method | Concentration ( |
Recovery (%) | RSD (%) | ||
---|---|---|---|---|---|
Taken | Added | Found ± SD | |||
Derivative spectrophotometric method | |||||
ESR | 1 | 0.3 | 1.36 ± 0.07 | 104.62 | 5.44 |
1.5 | 2.59 ± 0.06 | 103.60 | 2.24 | ||
3 | 3.67 ± 0.06 | 91.75 | 1.72 | ||
DRS | 2 | 0.3 | 2.30 ± 0.02 | 100.00 | 0.70 |
1.5 | 3.45 ± 0.16 | 98.57 | 4.55 | ||
3 | 4.82 ± 0.17 | 96.40 | 3.52 | ||
HPLC method | |||||
ESR | 0.5 | 0.3 | 0.79 ± 0.06 | 98.75 | 7.72 |
1.5 | 1.99 ± 0.04 | 99.50 | 1.96 | ||
3 | 3.73 ± 0.05 | 106.57 | 1.23 | ||
DRS | 1 | 0.2 | 1.12 ± 0.03 | 93.33 | 2.77 |
0.7 | 1.63 ± 0.03 | 95.88 | 1.96 | ||
1 | 1.93 ± 0.02 | 96.50 | 1.03 |
To examine the stability of the ESR and DRS solutions, the compounds in the mixture stored in the refrigerator at +4°C for a month and in the dark for 4 days at room temperature and then were analyzed in three replicates by the proposed methods under the selected conditions. The analyses results of these samples were compared with the results of freshly prepared drug solutions and found to be stable under these conditions.
The proposed methods were administered for the analysis of the drugs studied in their tablet form, namely, Angeliq, which contains 1 mg ESR and 2 mg DRS per tablet. For the first derivative spectrophotometric method, the mean recovery values were 101% (RSD% = 0.79) and 99% (RSD% = 0.03) for ESR and DRS, respectively (Table
Analysis of ESR and DRS in Angeliq tablets by developed methods (1 mg ESR and 2 mg DRS per tablet),
Statistical values | Derivative spectrophotometric method | HPLC method | Derivative spectrophotometric method | HPLC method |
---|---|---|---|---|
ESR | DRS | |||
Mean (mg) ± SD | 1.01 ± 0.008 | 0.97 ± 0.002 | 1.98 ± 0.000 | 1.96 ± 0.007 |
Recovery (%) | 101 | 97 | 99 | 98 |
RSD (%) | 0.79 | 0.21 | 0.03 | 0.36 |
In this study, a simple, rapid, accurate, and sensitive first derivative spectrophotometric and an RP-HPLC method were developed and validated for the simultaneous determination of ESR and DRS in their tablets for the first time. The HPLC method has a shorter analytical run. Both methods are cost effective compared to the LC-MS methods. Considering the linearity values and LOD values of DRS, the both proposed methods were more sensitive than reported RP-HPLC methods for the assay of the drug alone [
The authors report no conflict of interests.
Scientific Research Projects Coordination Unit of Istanbul University supported this work by the Projects (BYP-37557 and UDP-46099).